Application of marker in preparation of kit for diagnosing bronchial asthma or diagnosing and distinguishing bronchial asthma and chronic obstructive pulmonary disease

Abstract

The invention discloses application of a marker in preparation of a kit for diagnosing bronchial asthma or diagnosing and distinguishing bronchial asthma and chronic obstructive pulmonary disease, and belongs to the field of disease detection and diagnosis. The invention discloses a method. 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphorylcholine, beta-acetyl-gamma-o-alkyl-L-alpha-phosphatidylcholine and (R) Any one or two or all three of the-3-hydroxytetradecanoic acid and the-3-hydroxytetradecanoic acid have the value of diagnosing and distinguishing the chronic obstructive pulmonary disease and the bronchial asthma or diagnosing and distinguishing the bronchial asthma and healthy subjects. Therefore, a diagnostic kit for diagnosing and distinguishing the chronic obstructive pulmonary disease and the bronchial asthma or diagnosing and distinguishing the bronchial asthma can be prepared on the basis of the scheme, and the kit contains a compound standard substance corresponding to the scheme and can also contain an internal standard compound for mass spectrum quantitative analysis.

Description

Technical Field

[0001] This invention belongs to the field of disease detection and diagnosis, and relates to the application of disease diagnostic biomarkers, specifically the application of a biomarker in the preparation of a reagent kit for diagnosing bronchial asthma or diagnosing bronchial asthma and chronic obstructive pulmonary disease. Background Technology

[0002] Bronchial asthma (asthma) and chronic obstructive pulmonary disease (COPD) are two chronic respiratory diseases with high global morbidity and mortality, seriously affecting the health and lives of hundreds of millions of patients worldwide. Asthma is characterized by airway hyperresponsiveness and variable airflow limitation, often accompanied by an allergic inflammatory background, while COPD is characterized by irreversible airflow limitation, small airway remodeling, and emphysema, and its onset is often related to factors such as smoking and occupational exposure to harmful particles. Although the etiologies, inflammatory mechanisms, and treatment strategies of the two are significantly different, in actual clinical practice, due to the overlap in symptoms, changes in lung function, and some structural changes, accurate diagnosis of asthma and effective differentiation between asthma and COPD remain challenging.

[0003] Currently, the diagnosis of asthma mainly relies on typical clinical symptoms and pulmonary function tests showing variable airflow limitation, but this method has certain limitations:

[0004] 1. High symptom dependence and lack of specificity: Asthma symptoms highly overlap with many diseases, and cannot be distinguished by symptoms alone.

[0005] 2. Limited limitations of pulmonary function reversibility tests: Bronchodilator tests have limited sensitivity and are prone to false negatives; bronchial provocation tests, as the gold standard, have high operational requirements, many contraindications, and are risky; peak expiratory flow monitoring has low feasibility and poor result stability.

[0006] Accurate differentiation between asthma and COPD is crucial for developing appropriate treatment plans and assessing disease prognosis. Current diagnostic methods primarily include clinical symptom and medical history assessment, pulmonary function testing, and inflammatory biomarker detection. However, existing diagnostic methods have certain limitations:

[0007] 1. Overlapping clinical phenotypes and high subjectivity: Asthma and COPD symptoms and medical history highly overlap, especially in middle-aged and elderly patients where the characteristics are blurred. Differential diagnosis relies heavily on the doctor's experience, and the risk of misdiagnosis is high.

[0008] 2. Limited differentiation efficacy of pulmonary function indicators: As the gold standard, pulmonary function tests have insufficient sensitivity in the early stages of disease and poor repeatability, making it difficult to reliably distinguish between the two diseases.

[0009] 3. Insufficient specificity and universality of inflammatory markers: Commonly used biomarkers such as exhaled nitric oxide (FeNO) and blood eosinophils are easily affected by various factors, and the diagnostic efficacy of a single indicator is limited, making it difficult to accurately distinguish complex phenotypes.

[0010] 4. Lack of accurate and convenient objective diagnostic tools: Existing diagnostic methods are either invasive, expensive, or difficult to standardize, and are not suitable for large-scale screening. Clinical practice urgently needs high-precision, non-invasive, and easy-to-promote differential diagnostic methods.

[0011] Therefore, existing diagnostic criteria for asthma and methods for differentiating asthma from COPD fall short of clinical needs in terms of specificity, sensitivity, repeatability, and operability. With the development of precision medicine, there is an urgent clinical need for novel molecular diagnostic tools that are more objective, sensitive, and biologically grounded to compensate for the shortcomings of existing methods and improve the diagnostic efficacy of asthma and the ability to differentiate between asthma and COPD.

[0012] To overcome the shortcomings of the existing technology, this invention is proposed. Summary of the Invention

[0013] The purpose of this invention is to overcome the shortcomings of the prior art and provide a biomarker for use in the preparation of a kit for diagnosing bronchial asthma or diagnosing bronchial asthma and chronic obstructive pulmonary disease.

[0014] The above-mentioned objective of this invention is achieved through the following technical solution:

[0015] The use of a compound combination for the preparation of a diagnostic kit to differentiate between chronic obstructive pulmonary disease and bronchial asthma, the compound combination comprising 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid.

[0016] Preferably, the compound combination further includes an internal standard compound for quantitative mass spectrometry analysis.

[0017] More preferably, the internal standard compound is N-[(1S,2R,3E)-2-hydroxy-1-(hydroxymethyl)-3-heptadecene-1-yl]pentadecanamide and oleic acid-d9.

[0018] The use of a compound combination for preparing a diagnostic kit to differentiate between chronic obstructive pulmonary disease and bronchial asthma, the compound combination comprising any two or one of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid.

[0019] Preferably, the compound combination further includes an internal standard compound for quantitative mass spectrometry analysis.

[0020] More preferably, the internal standard compound is N-[(1S,2R,3E)-2-hydroxy-1-(hydroxymethyl)-3-heptadecene-1-yl]pentadecanamide and oleic acid-d9.

[0021] The use of a compound combination for preparing a kit for diagnosing bronchial asthma, the compound combination comprising 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid.

[0022] Preferably, the compound combination further includes an internal standard compound for quantitative mass spectrometry analysis.

[0023] More preferably, the internal standard compound is N-[(1S,2R,3E)-2-hydroxy-1-(hydroxymethyl)-3-heptadecene-1-yl]pentadecanamide and oleic acid-d9.

[0024] The use of a compound combination for preparing a kit for diagnosing bronchial asthma, the compound combination comprising any two or one of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid.

[0025] Preferably, the compound combination further includes an internal standard compound for quantitative mass spectrometry analysis.

[0026] More preferably, the internal standard compound is N-[(1S,2R,3E)-2-hydroxy-1-(hydroxymethyl)-3-heptadecene-1-yl]pentadecanamide and oleic acid-d9.

[0027] Beneficial effects:

[0028] 1. This invention has found that β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphatecholine, or (R)-3-hydroxytetradecanoic acid, when used alone, have high accuracy in distinguishing healthy subjects from asthma patients, all exceeding 95%. Therefore, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphatecholine, and (R)-3-hydroxytetradecanoic acid all possess value for individual diagnostic differentiation between healthy subjects and asthma patients.

[0029] 2. This invention has found that β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, or (R)-3-hydroxytetradecanoic acid, when used alone, have accuracy rates of 98%, 68%, and 83% in distinguishing between COPD patients and asthma patients, respectively. Those skilled in the art should know that a significant portion of commonly used clinical diagnostic markers have an accuracy rate of approximately 65% ​​to 75%. Therefore, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid all possess value for diagnosing and distinguishing between COPD patients and asthma patients when used alone.

[0030] 3. This invention has found that the combined diagnostic accuracy of any two of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid in differentiating between healthy subjects and asthma patients is high, all exceeding 90%. Therefore, the combination of any two of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid is valuable for diagnosing and differentiating between healthy subjects and asthma patients.

[0031] 4. This invention has found that the combined diagnostic accuracy of any two of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid in differentiating between COPD and asthma patients is high, exceeding 80%. Therefore, the combination of any two of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid is valuable for differentiating between COPD and asthma patients.

[0032] 5. This invention has found that the combined diagnostic accuracy of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid in distinguishing healthy subjects from asthma patients is as high as 96.2%. Therefore, the combination of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid is valuable for diagnosing and distinguishing healthy subjects from asthma patients.

[0033] 6. This invention has found that the combined diagnostic accuracy of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid in differentiating between COPD and asthma patients is as high as 97%. Therefore, the combination of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid is valuable for diagnosing and differentiating between COPD and asthma patients. Attached Figure Description

[0034] Figure 1 ROC curves for β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine to differentiate between asthma patients and healthy subjects or COPD patients and asthma patients: where: A represents asthma patients VS healthy subjects, B represents asthma patients VS COPD patients;

[0035] Figure 2 ROC curves for distinguishing between asthma patients and healthy subjects or COPD patients and asthma patients alone using 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine: where A represents asthma patients vs. healthy subjects, and B represents asthma patients vs. COPD patients.

[0036] Figure 3 ROC curves for the diagnosis of (R)-3-hydroxytetradecanoic acid alone, distinguishing between asthma patients and healthy subjects or between COPD patients and asthma patients: where: A represents asthma patients vs. healthy subjects, and B represents asthma patients vs. COPD patients.

[0037] Figure 4 ROC curves for the combined diagnosis of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine to differentiate healthy subjects from asthmatic patients;

[0038] Figure 5 ROC curves for the combined diagnosis of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine to differentiate between COPD and asthma patients.

[0039] Figure 6 ROC curves for the combined diagnosis of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid to differentiate healthy subjects from asthmatic patients;

[0040] Figure 7ROC curves for the combined diagnosis of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid to differentiate between COPD and asthma patients;

[0041] Figure 8 ROC curves for the combined diagnosis of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine and (R)-3-hydroxytetradecanoic acid to differentiate healthy subjects from asthmatic patients;

[0042] Figure 9 ROC curves for the combined diagnosis of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine and (R)-3-hydroxytetradecanoic acid to differentiate between COPD and asthma patients;

[0043] Figure 10 ROC curves for the combined diagnosis of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine and (R)-3-hydroxytetradecanoic acid to differentiate healthy subjects from asthmatic patients;

[0044] Figure 11 ROC curves for the combined diagnosis of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine and (R)-3-hydroxytetradecanoic acid to differentiate between COPD and asthma patients. Detailed Implementation

[0045] The substantive content of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should know that the scope of protection of the present invention should not be limited to these specific embodiments.

[0046] Example 1: Diagnostic efficacy of target biomarkers in diagnosing asthma or differentiating between asthma and COPD

[0047] I. Experimental Samples

[0048] Training set samples: Serum samples were collected from the First, Second and Third Affiliated Hospitals of Henan University of Traditional Chinese Medicine, Jiangsu Provincial Hospital of Traditional Chinese Medicine, Neixiang County People's Hospital, Songxian County People's Hospital, and Zhumadian Central Hospital. The samples consisted of 165 healthy subjects, 125 patients with COPD, and 99 patients with asthma, selected according to strict screening and exclusion criteria.

[0049] Validation set sample: Serum samples were collected from the First and Second Affiliated Hospitals of Henan University of Traditional Chinese Medicine, Jiangsu Provincial Hospital of Traditional Chinese Medicine, Neixiang County People's Hospital, Songxian County People's Hospital, Zhumadian Central Hospital, and the Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine. The samples consisted of 72 healthy subjects, 67 patients with COPD, and 33 patients with asthma, selected according to strict screening and exclusion criteria.

[0050] The inclusion criteria for healthy subjects and patients with diseases are as follows:

[0051] Healthy subjects: No history of cardiovascular, respiratory, liver, kidney, gastrointestinal, endocrine, hematological, mental, or nervous system diseases, or any of the above-mentioned diseases; no acute or chronic diseases; no history of drug allergies; and clinical laboratory test results within the normal reference range at the time of screening.

[0052] For patients with COPD: According to the Global Initiative for Chronic Obstructive Lung Disease (2025), the ratio of forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) after using a bronchodilator is < 0.7.

[0053] Asthma patients: According to the "Guidelines for the Prevention and Treatment of Bronchial Asthma" (2024), patients with recurrent wheezing, shortness of breath, chest tightness, cough and other symptoms, and who also meet one of the following three criteria, are diagnosed as asthma patients: (1) The peak expiratory flow rate in the bronchodilator test is ≥ 20% higher than the baseline; (2) The change in FEV1 between two visits is ≥ 12% and the absolute value is ≥ 200 ml; all of the above methods must exclude respiratory tract infection within 4 weeks before the examination; (3) Patients whose forced expiratory volume in one second is ≥ 80% of the predicted value, have small airway dysfunction in their pulmonary ventilation function at baseline, or have a change in FEV1 in the bronchodilator test of ≥ 10% and FeNO ≥ 35 ppb.

[0054] II. Experimental Instruments and Reagents

[0055] HPLC-grade methanol and isopropanol, HPLC-grade ammonium acetate, and LC-MS-grade acetonitrile and formic acid were purchased from Thermo Fisher Scientific, Shanghai, China; (R)-3-hydroxytetradecanoic acid (CAS No.: 28715-21-1, purity > 99%) was purchased from Avanti Biotechnology, USA; β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine (CAS No.: 65154-06-5, purity > 98%) was purchased from Chengdu Desite Biotechnology Co., Ltd.; 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine (CAS No.: 89947-79-5, purity > 99%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; ultrapure water was prepared using a laboratory Milli-Q ultrapure water purification system and purchased from Merck Millipore, Shanghai, China; a high-speed benchtop refrigerated centrifuge and a centrifugal concentrator were purchased from Thermo Fisher Scientific, USA. Scientific Inc.; the electronic balance was purchased from Mettler Toledo Instruments (Shanghai) Co., Ltd.

[0056] III. Experimental Methods

[0057] 1. Collection and processing of serum samples

[0058] Fasting peripheral blood was collected from the patient in the morning and placed in a test tube without anticoagulant. The blood was allowed to coagulate naturally at room temperature for 30 minutes. After the blood had coagulated, it was centrifuged at 3000 rpm for 10 minutes. The clear serum liquid at the top was carefully aspirated into a sterile lyophilized tube, labeled, and stored at -80°C for later use.

[0059] 2. Determination of target biomarker content in serum by UHPLC-QQQ MS

[0060] Testing instrument: SCIEX Triple Quad 6500 LC-MS / MS purchased from AB Sciex LLC, USA.

[0061] HPLC conditions: Column: ACQUITY UPLC® BEH C8 1.7 μm (2.1×100 mm Column), column temperature: 55 ℃, mobile phase A: 8 mol / L ammonium acetate + (6:4) acetonitrile / water, mobile phase B: 8 mol / L ammonium acetate + (9:1) isopropanol / acetonitrile, flow rate: 0.26 mL / min, injection volume: 2 μL, gradient elution program: 32% B (0-1.5 min), 32% B~85% B (1.5-15.5 min), 85% B~97% B (15.5-15.6 min), 97% B (15.6-18 min), 97% B~32% B (18-18.1 min), 32% B (18.1-20 min).

[0062] Mass spectrometry conditions: Electrospray ionization source, positive ion mode at 5500 V, ion source auxiliary gas 1 and ion source auxiliary gas 2 both at 55 Psi, curtain gas at 35 psi. Ion source temperature at 450 ℃. Multiple reaction monitoring (MRM) scanning mode was used, with a run time of 20 min.

[0063] Sample processing: 80 μL of serum from each patient was added to a 2 mL EP tube containing 300 μL of cold methanol. 10-20 μL was used to prepare a QC sample. The sample was vortexed, and 1 mL of methyl tert-butyl ether (containing oleic acid-d9 and N-[(1S,2R,3E)-2-hydroxy-1-(hydroxymethyl)-3-heptadecen-1-yl]pentadecanamide as an internal standard) was added. The extract was equilibrated at 4°C for 10 min, then centrifuged (18000 g, 4°C, 10 min). 1 mL of the supernatant was transferred to a 1.5 mL EP tube, concentrated under vacuum, and stored at -80°C for subsequent analysis. Before analysis, the sample was reconstituted in 50 μL of acetonitrile / isopropanol / water (65:30:5) containing 5 mM ammonium acetate.

[0064] 1-Palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, (R)-3-hydroxytetradecanoic acid, and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine standards were each dissolved to prepare a 1 mg / mL stock solution. 20 μL of each stock solution was used to prepare mixed standard solutions, which were then diluted to different concentrations. 20 μL of each concentration mixed standard solution was added to 60 μL of blank solvent and then transferred to a 2 mL EP tube containing 300 μL of cold methanol. 1 mL of methyl tert-butyl ether (containing internal standard) was added, and the mixture was vortexed. The extract was equilibrated at 4 °C for 10 min, then centrifuged (18000 g, 4 °C, 10 min). 1 mL of the supernatant was transferred to a 1.5 mL EP tube, concentrated under vacuum, and stored at -80 °C for subsequent analysis. Before analysis, the sample was reconstituted in 50 μL of acetonitrile / isopropanol / water (65:30:5) containing 5 mM ammonium acetate.

[0065] Standard curve establishment: MRM detection mode was used to detect mixed standard solutions of different concentrations. Data were integrated, and the deviation of the detected concentration was controlled within ±15% to exclude non-compliant mixed standard concentrations. Based on this, a standard curve was established, ensuring a correlation coefficient of ≥0.99. Sample testing: After sample reconstitution, samples from healthy subjects, COPD patients, and asthma patients were tested alternately to avoid interference between duplicate samples.

[0066] The target biomarkers in the samples were quantified using SCIEX OS 2.2 software (AB SCIEX).

[0067] 3. Data processing methods

[0068] In the training set, a difference of >1.2 and p<0.05 was considered statistically significant. For multiple target biomarkers, logistic regression was used to establish regression equations, generating a new variable logit[p]. ROC curve analysis was then performed on this new variable. In the validation set, the optimal cut-off value obtained from the ROC curve was used as the threshold to calculate the diagnostic accuracy of the combined biomarker analysis for COPD.

[0069] The principle of ROC curve evaluation method:

[0070] Basic evaluation indicators for diagnostic tests include sensitivity and specificity, while comprehensive evaluation indicators include the Youden index, ROC, and AUC. For evaluating a diagnostic test, it is first necessary to determine the true group of the sample being tested using the gold standard. For the disease group and healthy group determined by the gold standard, the results of the diagnostic test can be categorized as follows:

[0071] Positive (True Positive, TP); the diagnostic test result is positive (consistent with the gold standard result);

[0072] Negative (True Negative, TN); the diagnostic test result was negative (consistent with the gold standard result);

[0073] False positive (FP): A positive result in a diagnostic test (inconsistent with the gold standard result);

[0074] False negative (FN): A diagnostic test result is negative (inconsistent with the gold standard result).

[0075] This can be represented by Table 1:

[0076] Table 1

[0077]

[0078] The sensitivity of a diagnostic test = A / (A+C); the specificity of a diagnostic test = D / (B+D). Sensitivity and specificity determine the diagnostic sensitivity and specificity of a test relative to the gold standard. High sensitivity means fewer cases will be diagnosed as negative, resulting in a low rate of missed diagnoses; high specificity means fewer healthy cases will be diagnosed as positive, resulting in a low rate of false positives.

[0079] The ROC curve is a curve plotted based on the aforementioned sensitivity and specificity. Using the possible diagnostic cutoff values ​​in the diagnostic test as diagnostic points, the corresponding sensitivity and specificity are calculated according to the table above. Then, with sensitivity as the ordinate and 1-specificity as the abscissa, the sensitivity and specificity points at each diagnostic point are plotted on a coordinate graph. Connecting the coordinate points yields a smooth curve, which is the ROC curve. The more and denser the diagnostic points are, the smoother the resulting ROC curve will be.

[0080] The ROC curve uses each test result as a possible diagnostic cutoff value, and the area under the curve (AUC) indicates the accuracy of the diagnostic test. The AUC is widely accepted as an inherent accuracy indicator for evaluating the validity of diagnostic tests. An AUC of 0.5 indicates no diagnostic significance; an AUC between 0.5 and 0.7 indicates low diagnostic accuracy; an AUC between 0.7 and 0.9 indicates moderate diagnostic accuracy; and an AUC greater than 0.9 indicates high diagnostic accuracy.

[0081] IV. Experimental Results

[0082] 1. Differences in serum levels of the target biomarker among healthy subjects, COPD patients, and asthma patients

[0083] In the training set, as shown in Table 2, compared with healthy subjects, the serum levels of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine were significantly upregulated in asthmatic patients, while the levels of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid were significantly downregulated; as shown in Table 3, compared with patients with stable COPD, the serum levels of the above three markers were significantly decreased in asthmatic patients.

[0084] Table 2. Differences in serum levels of target biomarkers between healthy subjects and asthma patients.

[0085]

[0086] Table 3. Differences in serum levels of target biomarkers between COPD and asthma patients.

[0087]

[0088] 2. ROC curves for the diagnosis of asthma using target biomarkers alone or for differentiating between asthma and COPD.

[0089] ROC curve results for 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, (R)-3-hydroxytetradecanoic acid, and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine used alone to differentiate asthma patients from healthy subjects or to differentiate asthma patients from COPD patients are shown in Tables 4-5. Figures 1-3 As shown. Figures 1-3 In the diagram, A represents asthma patients versus healthy subjects, and B represents asthma patients versus COPD patients.

[0090] Table 4. ROC curves for distinguishing asthma patients versus healthy subjects using target biomarkers alone.

[0091]

[0092] Table 5. ROC curves for distinguishing between asthma patients and COPD patients using target biomarkers alone.

[0093]

[0094] ROC curve results showed that 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, (R)-3-hydroxytetradecanoic acid, and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, when used alone, demonstrated high accuracy in distinguishing between asthma patients and healthy subjects (AUC all above 0.9). β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, when used alone, also demonstrated high accuracy in distinguishing between asthma patients and COPD patients (AUC above 0.9), while 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine and (R)-3-hydroxytetradecanoic acid, when used alone, demonstrated moderate accuracy in distinguishing between asthma patients and COPD patients (AUC all between 0.7 and 0.9). Furthermore, the Wieden index was calculated based on the ROC curve coordinates: specificity + sensitivity - 1. The relative concentration of the biomarker corresponding to the maximum Wieden index was the optimal cut-off value for distinguishing between asthma patients and healthy subjects, or between asthma patients and COPD patients, as shown in Table 6.

[0095] Table 6. Optimal cut-off values ​​for target metabolites to distinguish between asthma vs. healthy subjects or between asthma and COPD.

[0096]

[0097] 3. ROC curves for diagnosing and differentiating asthma patients from healthy subjects using multiple target biomarkers, or for differentiating asthma patients from COPD patients.

[0098] 3.1 The combined use of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine to differentiate healthy subjects from asthmatic patients

[0099] Using the levels of two biomarkers in the training set samples as independent variables (let X1 = β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine content, X2 = 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine content), and the group (i.e., healthy subjects and asthma patients) as the dependent variable, logistic regression was performed on the levels of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine in the serum samples of healthy subjects and asthma patients, yielding the logistic regression equation: Logit[p] = -174.971502 + 0.011471 X1 - 0.446967 X2; substituting the biomarker content in each serum sample into this logistic regression equation, the regression value logit[p] for each serum sample can be obtained. Using the possible regression value logit[p] as the diagnostic point, sensitivity and specificity were calculated, and ROC curves were plotted accordingly (e.g., ...). Figure 4The AUC is 1, indicating high accuracy. The Wieden index is calculated based on the ROC curve coordinates: specificity + sensitivity - 1. The logit[p] value corresponding to the maximum Wieden index is the optimal cut-off value of 0.5001, which can effectively differentiate between healthy subjects and asthma patients.

[0100] 3.2 The combined use of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline to differentiate between COPD patients and asthma patients.

[0101] Using the levels of two biomarkers in the training set samples as independent variables (let X1 = β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, X2 = 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine content), and the group (i.e., COPD patients and asthma patients) as the dependent variable, logistic regression was performed on the levels of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine in the serum samples of COPD and asthma patients, yielding the logistic regression equation: Logit[p] = -18.525 - 0.005805 X1 - 3.060096 X2; substituting the biomarker content in each serum sample into this logistic regression equation, the regression value logit[p] for each serum sample can be obtained. Using the possible regression value logit[p] as the diagnostic point, sensitivity and specificity were calculated, and ROC curves were plotted accordingly (e.g., ...). Figure 5 The AUC is 1, indicating high accuracy. The Wieden index is calculated based on the ROC curve coordinates: specificity + sensitivity - 1. The logit[p] value corresponding to the maximum Wieden index is the optimal cut-off value of 0.4998, which can effectively differentiate between COPD patients and asthma patients.

[0102] 3.3 The combined use of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid to differentiate healthy subjects from asthmatic patients

[0103] Using the levels of two biomarkers in the training set samples as independent variables (let X1 = β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine content, X2 = (R)-3-hydroxytetradecanoic acid content), and the group (i.e., healthy subjects and asthma patients) as the dependent variable, logistic regression was performed on the levels of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid in the serum samples of healthy subjects and asthma patients, yielding the logistic regression equation: Logit[p] = -111.391245 + 0.004424 X1 + 0.546658 X2; substituting the biomarker levels in each serum sample into this logistic regression equation, the regression value logit[p] for each serum sample was obtained. Using the possible regression value logit[p] as the diagnostic point, sensitivity and specificity were calculated, and an ROC curve was plotted accordingly (e.g., ...). Figure 6 The AUC is 1, indicating high accuracy. The Wieden index is calculated based on the ROC curve coordinates: specificity + sensitivity - 1. The logit[p] value corresponding to the maximum Wieden index is the optimal cut-off value of 0.5, which can effectively differentiate between healthy subjects and asthma patients.

[0104] 3.4 The combined use of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid to differentiate between COPD patients and asthma patients

[0105] Using the levels of two biomarkers in the training set samples as independent variables (let X1 = β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine content, X2 = (R)-3-hydroxytetradecanoic acid content), and the group (i.e., COPD patients and asthma patients) as the dependent variable, logistic regression was performed on the levels of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid in the serum samples of COPD and asthma patients, yielding the logistic regression equation: Logit[p] = 9.132397 - 0.000394 X1 - 0.05245 X2; substituting the biomarker levels in each serum sample into this logistic regression equation, the regression value logit[p] for each serum sample was obtained. Using the possible regression value logit[p] as the diagnostic point, sensitivity and specificity were calculated, and ROC curves were plotted accordingly (e.g., ...). Figure 7 The AUC was 0.995, indicating high accuracy. The Wieden index was calculated based on the ROC curve coordinates: specificity + sensitivity - 1. The logit[p] value corresponding to the maximum Wieden index was the optimal cut-off value of 0.349, which is sufficient to differentiate between COPD patients and asthma patients.

[0106] 3.5 Combined Diagnostic Differentiation of Healthy Subjects vs. Asthma Patients Using 1-Palmitoyl-2-Glutamicyl-sn-glycerol-3-phosphate choline and (R)-3-hydroxytetradecanoic acid

[0107] Using the levels of two biomarkers in the training set samples as independent variables (let X1 = 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine content, X2 = (R)-3-hydroxytetradecanoic acid content), and the group (i.e., healthy subjects and asthma patients) as the dependent variable, logistic regression was performed on the levels of (R)-3-hydroxytetradecanoic acid and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine in the serum samples of healthy subjects and asthma patients, yielding the logistic regression equation: Logit[p] = -4.547486 + 0.004237 X1 + 0.090891 X2; substituting the biomarker levels in each serum sample into this logistic regression equation, the regression value logit[p] for each serum sample was obtained. Using the possible regression value logit[p] as the diagnostic point, sensitivity and specificity were calculated, and ROC curves were plotted accordingly (e.g., ...). Figure 8 The AUC was 0.984, indicating high accuracy. The Wieden index was calculated based on the ROC curve coordinates: specificity + sensitivity - 1. The logit[p] value corresponding to the maximum Wieden index was the optimal cut-off value of 0.951, which distinguishes healthy subjects from asthma patients.

[0108] 3.6 Combined Diagnosis of 1-Palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline and (R)-3-hydroxytetradecanoic acid to Differentiate Between COPD and Asthma Patients

[0109] Using the levels of two biomarkers in the training set samples as independent variables (let X1 = 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine content, X2 = (R)-3-hydroxytetradecanoic acid content), and the group (i.e., COPD patients and asthma patients) as the dependent variable, logistic regression was performed on the levels of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine and (R)-3-hydroxytetradecanoic acid in the serum samples of COPD and asthma patients, yielding the logistic regression equation: Logit[p] = 1.052109 - 0.001758 X1 - 0.047174 X2; substituting the biomarker levels in each serum sample into this logistic regression equation, the regression value logit[p] for each serum sample was obtained. Using the possible regression value logit[p] as the diagnostic point, sensitivity and specificity were calculated, and ROC curves were plotted accordingly (e.g., ...). Figure 9The AUC was 0.786, indicating high accuracy. The Wieden index was calculated based on the ROC curve coordinates: specificity + sensitivity - 1. The logit[p] value corresponding to the maximum Wieden index was the optimal cut-off value of 0.524, which is sufficient to differentiate between COPD patients and asthma patients.

[0110] 3.7 Combined diagnostic differentiation between healthy subjects and asthma patients using β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid.

[0111] Using the levels of three biomarkers in the training set samples as independent variables (let X1 = β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine content, X2 = 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine content, X3 = (R)-3-hydroxytetradecanoic acid content), and group (i.e., healthy subjects and asthma patients) as the dependent variable), logistic regression was performed on the levels of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, and (R)-3-hydroxytetradecanoic acid in the serum samples of healthy subjects and asthma patients, resulting in the logistic regression equation: Logit[p] = -46.797894 + 0.002177 X1 - 0.078095 X2 + 0.256346 X3; Substitute the biomarker content in each serum sample into the logistic regression equation to obtain the regression value logit[p] for each serum sample. Use the possible regression value logit[p] as the diagnostic point to calculate sensitivity and specificity, and plot the ROC curve accordingly (e.g., Figure 10 The AUC is 1, indicating high accuracy. The Wieden index is calculated based on the ROC curve coordinates: specificity + sensitivity - 1. The logit[p] value corresponding to the maximum Wieden index is the optimal cut-off value of 0.500, which can effectively differentiate between healthy subjects and asthma patients.

[0112] 3.8 Combined diagnosis and differentiation of COPD patients versus asthma patients using β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid.

[0113] Using the levels of three biomarkers in the training set samples as independent variables (let X1 = β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine content, X2 = 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine content, X3 = (R)-3-hydroxytetradecanoic acid content), and the group (i.e., COPD patients and asthma patients) as the dependent variable), logistic regression was performed on the levels of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, and (R)-3-hydroxytetradecanoic acid in the serum samples of COPD and asthma patients, resulting in the logistic regression equation: Logit[p] = -9.293404 - 0.00541 X 1+ 2.380658 X2 + 0.853733 X3; Substituting the biomarker content in each serum sample into this logistic regression equation, the regression value logit[p] for each serum sample can be obtained. Using the possible regression value logit[p] as the diagnostic point, sensitivity and specificity are calculated, and an ROC curve is plotted accordingly (e.g., Figure 11 The AUC is 1, indicating high accuracy. The Wieden index is calculated based on the ROC curve coordinates: specificity + sensitivity - 1. The logit[p] value corresponding to the maximum Wieden index is the optimal cut-off value of 0.4999, which can effectively differentiate between COPD and asthma patients.

[0114] 4. Validate the accuracy of single target biomarker in differentiating healthy subjects from asthma patients.

[0115] In the validation set, the optimal cut-off value for distinguishing healthy subjects from asthma patients using β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, or (R)-3-hydroxytetradecanoic acid as the diagnostic threshold was used to predict serum samples from healthy subjects and asthma patients. Serum samples with β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine or (R)-3-hydroxytetradecanoic acid levels above the diagnostic threshold were predicted to be healthy subjects, while those below the threshold were predicted to be asthma patients. Similarly, serum samples with 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine levels above the diagnostic threshold were predicted to be asthma patients, while those below the threshold were predicted to be healthy subjects. The accuracy of this target biomarker in distinguishing healthy subjects from asthma patients was calculated by dividing the number of correctly predicted samples by the total number of samples (10⁵). The diagnostic accuracy of each target biomarker is shown in Table 7.

[0116] Table 7. Accuracy of single-target metabolite diagnosis in distinguishing healthy subjects from asthma patients.

[0117]

[0118] 5. Validate the accuracy of a single target biomarker in distinguishing between COPD patients and asthma patients.

[0119] In the validation set, the optimal cut-off value for distinguishing COPD patients from asthma patients using β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, or (R)-3-hydroxytetradecanoic acid as the diagnostic threshold was used to predict serum samples from COPD and asthma patients. Serum samples with β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine or (R)-3-hydroxytetradecanoic acid levels above the diagnostic threshold were predicted to be COPD patients, while those below the threshold were predicted to be asthma patients. Similarly, serum samples with 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine levels above the diagnostic threshold were predicted to be asthma patients, while those below the threshold were predicted to be COPD patients. The accuracy of this target biomarker in distinguishing COPD vs. asthma patients was calculated by dividing the number of correctly predicted samples by the total number of samples (100). The diagnostic accuracy of each target biomarker is shown in Table 8.

[0120] Table 8. Accuracy of single target metabolite diagnosis in differentiating COPD patients from asthma patients.

[0121]

[0122] 6. Validate the accuracy of combined diagnosis of multiple target biomarkers in differentiating between asthma patients and healthy subjects, or in differentiating between asthma patients and COPD patients.

[0123] 6.1 Accuracy of combined diagnosis of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline in distinguishing between healthy subjects and asthmatic patients

[0124] In the validation set, the levels of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine in serum samples from healthy subjects and asthma patients were substituted into the logistic regression equation that distinguishes healthy subjects from asthma patients using β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine. The regression value logit[p] for each sample was calculated. The optimal cut-off value for the combined use of acyl-2-glutaryl-sn-glycerol-3-phosphocholine to distinguish between healthy subjects and asthmatic patients is the diagnostic threshold. Logit[p] above this diagnostic threshold is predicted as a healthy subject, and below this threshold is predicted as an asthmatic patient. The accuracy of the combined use of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine to distinguish between healthy subjects and asthmatic patients is calculated by dividing the number of correctly predicted samples by the total number of samples (10² / 10⁵), which is 97.1%.

[0125] 6.2 Accuracy of combined β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline in differentiating COPD patients from asthma patients.

[0126] In the validation set, the levels of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine in serum samples from COPD and asthma patients were substituted into the logistic regression equation for distinguishing COPD patients from asthma patients using β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine. The regression value logit[p] for each sample was calculated. The optimal cut-off value for palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine in distinguishing between COPD patients and asthma patients is the diagnostic threshold. Logit[p] above the diagnostic threshold is predicted to indicate asthma patients, and below the diagnostic threshold is predicted to indicate COPD patients. The accuracy of the combined use of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine in distinguishing between COPD patients and asthma patients is calculated by dividing the number of correctly predicted samples by the total number of samples (98 / 100). The accuracy is 98%.

[0127] 6.3 Accuracy of combined β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid in distinguishing healthy subjects from asthma patients

[0128] In the validation set, the levels of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid in serum samples from healthy subjects and asthma patients were substituted into the logistic regression equation for distinguishing between healthy subjects and asthma patients using β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid. The regression value logit[p] for each sample was calculated. The optimal cut-off value for distinguishing between healthy subjects and asthma patients using β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid was used as the diagnostic threshold. Logit[p] values ​​higher than the diagnostic threshold were predicted to be those of healthy subjects, while logit[p] values ​​lower than the diagnostic threshold were predicted to be those of asthma patients. The accuracy of distinguishing between healthy subjects and asthma patients using β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid was calculated by dividing the number of correctly predicted samples by the total number of samples (101 / 105). The accuracy was 96.2%.

[0129] 6.4 Accuracy of combined diagnosis of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid in differentiating between COPD and asthma patients

[0130] In the validation set, the levels of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid in serum samples from COPD and asthma patients were substituted into the logistic regression equation for distinguishing COPD patients from asthma patients using β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid. The regression value logit[p] for each sample was calculated. The optimal cut-off value for the combined use of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid to distinguish between COPD patients and asthma patients is the diagnostic threshold. Logit[p] above this diagnostic threshold is predicted to indicate asthma patients, and below this threshold is predicted to indicate COPD patients. The accuracy of the combined use of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid to distinguish between COPD patients and asthma patients is 99%, which is calculated by dividing the number of correctly predicted samples by the total number of samples (99 / 100).

[0131] 6.5 Accuracy of combined diagnosis of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline and (R)-3-hydroxytetradecanoic acid in differentiating healthy subjects from asthma patients

[0132] In the validation set, the levels of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline and (R)-3-hydroxytetradecanoic acid in serum samples from healthy subjects and asthma patients were substituted into the logistic regression equation for distinguishing between healthy subjects and asthma patients using 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline and (R)-3-hydroxytetradecanoic acid. The regression value logit[p] for each sample was calculated. The optimal cut-off value for the combined use of (R)-3-hydroxytetradecanoic acid to distinguish between healthy subjects and asthma patients is the diagnostic threshold. Logit[p] above this diagnostic threshold is predicted as a healthy subject, and below this diagnostic threshold is predicted as an asthma patient. The accuracy of the combined use of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine and (R)-3-hydroxytetradecanoic acid to distinguish between healthy subjects and asthma patients is calculated by dividing the number of correctly predicted samples by the total number of samples (98 / 105). The accuracy is 93.3%.

[0133] 6.6 The accuracy of combined diagnosis of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline and (R)-3-hydroxytetradecanoic acid in differentiating COPD patients from asthma patients.

[0134] In the validation set, the levels of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline and (R)-3-hydroxytetradecanoic acid in serum samples from COPD and asthma patients were substituted into the logistic regression equation for distinguishing COPD patients from asthma patients using 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline and (R)-3-hydroxytetradecanoic acid. The regression value logit[p] for each sample was calculated. The 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline content was then used as the basis for further analysis. The optimal cut-off value for distinguishing between COPD patients and asthma patients by combining 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline and (R)-3-hydroxytetradecanoic acid is the diagnostic threshold. A logit[p] above the diagnostic threshold is predicted to indicate asthma, while a logit[p] below the diagnostic threshold is predicted to indicate COPD. The accuracy of distinguishing between COPD patients and asthma patients by combining 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline and (R)-3-hydroxytetradecanoic acid is 81%.

[0135] 6.7 Accuracy of combined diagnosis of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, (R)-3-hydroxytetradecanoic acid, and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine in differentiating healthy subjects from asthmatic patients

[0136] In the validation set, the levels of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, (R)-3-hydroxytetradecanoic acid, and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine in serum samples from healthy subjects and asthma patients were substituted into the logistic regression equation that jointly distinguishes healthy subjects from asthma patients. The regression value logit[p] for each sample was calculated. The above-mentioned 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, (R)-3-hydroxytetradecanoic acid, and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine were used to differentiate between healthy subjects and asthma patients. The optimal cut-off value for distinguishing between healthy subjects and asthma patients using (R)-3-hydroxytetradecanoic acid and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine is the diagnostic threshold. Logit[p] above this diagnostic threshold is predicted as a healthy subject, and below this threshold is predicted as an asthma patient. The accuracy of distinguishing between healthy subjects and asthma patients using 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine, (R)-3-hydroxytetradecanoic acid, and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine is 96.2%, calculated by dividing the number of correctly predicted samples by the total number of samples (101 / 105).

[0137] 6.8 The accuracy of combined diagnosis of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, (R)-3-hydroxytetradecanoic acid, and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine in differentiating between COPD and asthma patients.

[0138] In the validation set, the levels of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, (R)-3-hydroxytetradecanoic acid, and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine in serum samples from COPD and asthma patients were substituted into the logistic regression equation for the combined differentiation of COPD and asthma patients. The regression value logit[p] for each sample was calculated, and the above-mentioned 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline was used as the basis for further analysis. The optimal cut-off value for distinguishing between COPD patients and asthma patients using the combination of (R)-3-hydroxytetradecanoic acid and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine was the diagnostic threshold. Logit[p] above this diagnostic threshold was predicted as an asthma patient, and below this threshold was predicted as a COPD patient. The accuracy of distinguishing between COPD patients and asthma patients using the combination of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, (R)-3-hydroxytetradecanoic acid and β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine was 97%, calculated by dividing the number of correctly predicted samples by the total number of samples (97 / 100).

[0139] In summary:

[0140] 1. Example 1 experiments showed that β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, or (R)-3-hydroxytetradecanoic acid, when used alone, demonstrated high accuracy in distinguishing healthy subjects from asthma patients, all exceeding 95%. Therefore, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid all possess value for individual diagnostic differentiation between healthy subjects and asthma patients.

[0141] 2. Example 1 experiments found that β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, or (R)-3-hydroxytetradecanoic acid alone had accuracy rates of 98%, 68%, and 83% in distinguishing between COPD patients and asthma patients, respectively. Those skilled in the art should know that a significant portion of commonly used clinical diagnostic markers have an accuracy of approximately 65%–75%. Therefore, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid all possess value for individual diagnosis in distinguishing between COPD patients and asthma patients.

[0142] 3. Example 1 experiments showed that the combined diagnostic accuracy of any two of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid in differentiating healthy subjects from asthma patients was high, all exceeding 90%. Therefore, any combination of any two of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid is valuable for diagnosing and differentiating healthy subjects from asthma patients.

[0143] 4. Example 1 experiments showed that the combined diagnostic accuracy of any two of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid in differentiating between COPD and asthma patients was high, all exceeding 80%. Therefore, the combination of any two of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid is valuable for differentiating between COPD and asthma patients.

[0144] 5. Example 1 experiment found that the combined diagnostic accuracy of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid in distinguishing healthy subjects from asthma patients was as high as 96.2%. Therefore, the combination of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid is valuable for diagnosing and distinguishing healthy subjects from asthma patients.

[0145] 6. Example 1 experiment found that the combined diagnostic accuracy of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid in differentiating COPD patients from asthma patients reached 97%. Therefore, the combination of β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphate choline, and (R)-3-hydroxytetradecanoic acid is valuable for diagnosing and differentiating COPD patients from asthma patients.

[0146] Example 2: Diagnostic Kit

[0147] 1. An asthma diagnostic kit for diagnosing and differentiating asthma patients from healthy subjects, the kit containing a standard of the diagnostic markers β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, or (R)-3-hydroxytetradecanoic acid.

[0148] 2. An asthma diagnostic kit for diagnosing and differentiating asthma patients from healthy subjects, the kit containing standards of any two of the diagnostic markers β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine and (R)-3-hydroxytetradecanoic acid.

[0149] 3. An asthma diagnostic kit for diagnosing and differentiating asthma patients from healthy subjects, the kit containing diagnostic markers β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine and (R)-3-hydroxytetradecanoic acid standards.

[0150] 4. A diagnostic kit for differentiating between patients with COPD and asthma, the kit containing a standard of the diagnostic markers β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphatecholine, or (R)-3-hydroxytetradecanoic acid.

[0151] 5. A diagnostic kit for differentiating between patients with COPD and asthma, the kit containing standards of any two of the diagnostic markers β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine and (R)-3-hydroxytetradecanoic acid.

[0152] 6. A diagnostic kit for differentiating between patients with COPD and asthma, the kit containing standards of the diagnostic markers β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine, 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphatecholine and (R)-3-hydroxytetradecanoic acid.

[0153] The diagnostic kits described above may also contain solvents (such as methanol) for extracting biomarkers from serum and internal standards (such as oleic acid-d9 and N-[(1S,2R,3E)-2-hydroxy-1-(hydroxymethyl)-3-heptadecene-1-yl]pentadecanamide) for mass spectrometry quantitative analysis; for ease of quantification, each standard and internal standard is packaged separately.

[0154] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. The use of a compound combination for preparing a diagnostic kit to differentiate between chronic obstructive pulmonary disease and bronchial asthma, the compound combination comprising 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid.

2. The use according to claim 1, wherein the compound combination further comprises an internal standard compound for quantitative mass spectrometry analysis.

3. The use according to claim 2, wherein the internal standard compound is N-[(1S,2R,3E)-2-hydroxy-1-(hydroxymethyl)-3-heptadecen-1-yl]pentadecanamide and oleic acid-d9.

4. Use of a compound combination for preparing a diagnostic kit for differentiating chronic obstructive pulmonary disease from bronchial asthma, the compound combination comprising any two or one of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid.

5. The use according to claim 4, wherein the compound combination further comprises an internal standard compound for quantitative mass spectrometry analysis.

6. The use according to claim 5, wherein the internal standard compound is N-[(1S,2R,3E)-2-hydroxy-1-(hydroxymethyl)-3-heptadecen-1-yl]pentadecanamide and oleic acid-d9.

7. Use of a compound combination for preparing a kit for diagnosing bronchial asthma, the compound combination comprising 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid.

8. The use according to claim 7, wherein the compound combination further comprises an internal standard compound for quantitative mass spectrometry analysis.

9. Use of a compound combination for preparing a kit for diagnosing bronchial asthma, the compound combination comprising any two or one of 1-palmitoyl-2-glutaryl-sn-glycerol-3-phosphocholine, β-acetyl-γ-O-alkyl-L-α-phosphatidylcholine and (R)-3-hydroxytetradecanoic acid.

10. The use according to claim 9, wherein the compound combination further comprises an internal standard compound for quantitative mass spectrometry analysis.

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